AU2008278056B2

AU2008278056B2 - New antimicrobial compounds, their synthesis and their use for treatment of mammalian infections

Info

Publication number: AU2008278056B2
Application number: AU2008278056A
Authority: AU
Inventors: Vadim Makarov; Ute Mollmann; T. Cole Stewart
Original assignee: Leibniz Institut fuer Naturstoff Forschung und Infektionsbiol eVi
Current assignee: Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie Ev Hans-Knoll-Institut
Priority date: 2007-07-16
Filing date: 2008-06-25
Publication date: 2013-09-26
Anticipated expiration: 2028-06-25
Also published as: CA2693088C; WO2009010163A1; AU2008278056A1; JP2010533661A; ATE482939T1; EA201070145A1; MA31607B1; CN101809009B; KR101588064B1; CO6321160A2; ZA201000491B; MX2010000672A; UA99300C2; CN101809009A; DE602008002843D1; JP5502731B2; CA2693088A1; TN2010000023A1; US8440842B2; EP2020406A1

Abstract

The present invention relates to new antimicrobial compounds, their synthesis and their use for treatment of mammalian infections The present invention aims at the generation of new compounds with activity against

Description

-1l New antimicrobial compounds, their synthesis and their use for treatment of mammalian infections Description 5 The present invention relates generally to novel benzothiazin derivatives and their use as antibacterial agents in infectious diseases of mammals (humans and animals) caused by bacteria, especially diseases like tuberculosis (TB) and leprosy caused by nycobacteria. Thiazinone, their derivatives and their use as antibacterial agents, 10 especially against mycobacteria (TB), laid open for public in AR 24 25 67 Al, AU 37 04 400 Al, CA 13 22 551 Cl or EP 0 245 901 B1 for instance. As known, there is a threatening worldwide increase in tuberculosis infections with tnycobacteria which developed resistance against the 15 available therapeutics (B.R.Bloom, J.L.Murray, Tuberculosis: commentary on a reemergent killer. Science 1992, 257, 1055-1064). Extremely dangerous is the development of multidrug resistant (MDR) inycobacteria. These are mycobacteria, resistant at least against two of the most active tuberculosis drugs, isoniazid and rifampicin, but also 20 against streptomycin, pyranzinamid and ethambutol. The proportion of MDR-TB in some countries is already more than 20%. Even more threatening became the situation since the first cases of XDR-TB (extremly resistant TB) were diagnosed last year in South Africa. Now XDR-TB is already spread over all continents. Mycobacteria causing 25 XDR-TB are resistant against the first line TB drugs Rifampicin, Isoniazid, Pyrazinamid, Ethambutol and additionally against the second line chinolones and aminoglycosides. (Nature Med. 2007,13, 295-298) Together with the increased number of TB diseases generally, worldwide it causes about 2.000.000 deaths annually. 30 For the treatment of such diseases, like (TB) or leprosy, there is an urgent need for new drugs with new mechanisms of actions, especially to overcome drug resistance and to overcome the known dramatic side effects of the available drugs.

-2 Object of the invention The present invention aims at the generation of new compounds with activity against imycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance and/or to 5 at least provide the public with a useful choice. Solution of the technical problem In one aspect the invention relates to compounds of the formula I RI R5

R

3 ,, S" N 'R 0 R6

R

4 0 (I) wherein R' and R 2 are, independently of each other, NO 2 , NR 7 R', 10 NHOR 9 , COOR 9 , CN, CONROR", CHO, F, Cl, Br, SO 2 NR1 2

R

3 , lower alkoxy, OCF 3 , mono-, di or trifluoromethyl;

R

3 and R4 are, independently of each other, I], a saturated or unsaturated, linear or branched aliphatic radical having 1-3 chain members, F, Cl, Br, lower alkoxy; 15 R 5 is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R is a radical: wherein X is saturated or unsaturated, halogenated or unhalogenated, 20 linear or branched aliphatic radical having 1-5 chain members, or

R

5 and R 6 together represent bivalent radicals wherein n is 1-4: -3 /-JNfCH2)n or4 R - R are, independently of each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical 5 having 1-5 chain members, phenyl, benzyl or R 7 and R 8 together, R' 0 and R" together, R1 2 and R1 3 together represent a linear or branched aliphatic bivalent radical having 1-7 chain members; R and R' 5 are, independently of each other, H, linear or branched aliphatic radical having 1-5 chain members, F, Cl, Br, NO 2 , NH 2 , CF 3 . 10 In another aspect the invention relates to the use of a compound of formula (I) or a salt thereof of the invention for the preparation of a pharmaceutical composition. In another aspect the invention relates to the use of a compound of fonnula (I) or a salt thereof of the invention for the preparation of a 15 medicament for the therapeutic or prophylactic treatment of microbial infection in mammals. In another aspect the invention relates to the pharmaceutical composition comprising a compound of the invention. In another aspect the invention relates to a compound of the invention or 20 a salt thereof for use in a method for the therapeutic or prophylactic treatment of microbial infection in mammals. In another aspect the invention relates to a compound of the invention or a salt thereof for use in a method for the therapeutic or prophylactic treatment of tuberculosis or leprosy infection in mammals. 25 In another aspect the invention relates to a method for the preparation of 2-NR'R 6 -4H-1,3-benzothiazin-4-ones according to formula (I) of the invention, comprising reacting 2-chlorobenzcarboxamides with 1.1 to - 3a 2.5 times in excess of metal salts of alkylxanthogenate at a temperature of 20-100'C in a solvent and subsequently treating the resulting 2 alkoxy-4H-1,3-benzothiazin-4-one with an amine HNR 5 R 6 in a solvent at a temperature of 20-100 C. 5 Certain statements that appear below are broader than what appears in the statements of the invention above. These statements are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which defines the scope of the invention. 10 In a preferred embodiment described are compounds of the formula (I) selected from the group consisting of 2-[4-(2-R 4 ,5-R"5-pheny1)piperazin- 1 -yl]-8-nitro-6-(trifluoromethyl)-4ff -1,3-benzothiazin-4-one, 2-[4-(2-R ,-"pey~ieai--l-~itru,-6-(trifluoromet-hy1)-4H1 -1,3-benzothiazin-4-onie, 2-[4-(3-R',5 -R' ;-pheny1)piperazin- 1 -yi]-8-nitro-6-(trifluoromethyl)-4H -1,3-benzothiazin-4-one, 2-[benzvl(nethy)amino]-8-nito-6-.R4H- 1,3-benzothiazin-4-one 2-[benzy1(R 5 )amino]-8-nitro-6-(trifluoronethyl)-4I!-1,3-benzothiazin-4 one, 2- [benzyl(R$)amino]-8-nitro- 6-R24H- 1,3-benzothiazin-4-one, 2-[benzyl(R5)amino]-8-nitro-6-chloro-4H-1,3-benzothiazin-4-one, 2-[benzyl(R 5 )amino]-8-nitro-6-fluoro-4H-1, 3-benzothiazin-4-one, 2-[benzyl(R)aminio]-8-R -6-(trifluorometbyl)-4H-1,3-benzotbiazi n-4 one, wherein R', R 5 , R1 4 and R 15 have the above meanings. The present invention is even more particularly concerned with at least one 15 compound selected from the group consisting of (followed by page 4) WO 2009/010163 PCT/EP2008/005142 -4 2-[4-(4-Chlorophenyl)piperazin- 1-yl]-6,8-dinitro-4H- 1,3 -benzothiazin-4 one, 2-[4-(5-chloro-2-methylphenyl)piperazin- 1-yl]-8-nitro-6-(trifluorome thyl)-4H- 1,3-benzothiazin-4-one, 5 8-Nitro-6-(trifluoromethyl)-2- {4-[3-(trifluoromethyl)phenyl]piperazin- 1 yl} -4H- 1,3-benzothiazin-4-one, 2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3 -benzothia zin-4-one, 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3 -benzothia 10 zin-4-one, 2-[4-(2-Fluorophenyl)piperazin- 1-yl]-8-nitro-6-(trifluoromethyl)-4H- 1,3 benzothiazin-4-one, 2-(4-Benzylpiperazin- 1-yl)-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzo thiazin-4-one, 15 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H- 1,3 -benzothiazin-4-one, 2- {Methyl[(1R)- 1 -phenylethyl] amino } -8-nitro-6-(trifluoromethyl)-4H 1,3 -benzothiazin-4-one, 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H- 1,3-benzothiazin-4-one. 20 We used 4 different methods for the synthesis of new and novel 1,3 benzothiazin-4-one derivatives. Methods A, B and C propose to use as starting material well known polysubstituted 2-chloro(bromo) benzcarboxamides, many of them described in the literature or can be easily prepared by analogues methods (Isaew S. G. Farm. Zh. (Kiev), 25 2000, 52; Makosza M., Nizamov S. Org. Prep. and Proced. Int., 1997, 29, 707; Nerin C., Tomes A. R., Domento C., Cacho J. J. Agr. and Food Chem., 1996, 44, 4009; Thiel W., Mayer R., Jauer E.-A., Modrow H., Dost H. J. Prakt. Chem., 1986, 328, 497; Yokoyama M., Yoshida S., Imamoto T. Synthesis, 1982, 591; Romanowski J., Eckstein Z. Pol. J 30 Chem., 1984, 58, 263; Nisato D., Sacilotto R., Frigerio M., Boveri S., Palmisano G., Lesma G. Org. Prep. Proced. Int., 1985, 17, 75; Oikawa N., Nakagawa Y., Nishimura K., Ueno T., Fujita T., Pestic. Sci., 1994, 41, 139; Welch D.E., Baron R.R., J. Med. Chem., 1969, 12, 299; Fuller R.W., Molloy B.B., Day W.A., Roush B.W., March M.M., J Med. 35 Chem., 1973, 16, 101 and many others).

WO 2009/010163 PCT/EP2008/005142 -5 R1 R3 Cl excess NaSC(S)NR 5

R

6 or addition light alkaly R2*CONH 2 R4 Method B Method A NaSC(S)NR 5

R

6 R1 R5 S R3 I R1 Ak R5 R2W R6 R3 S N R2 CONH 2 O R4 Method A The starting 2-chlorobenzcarboxamides were treated by 1,0-1,2 5 equimolar quantity of the metal salts of dithiocarbamates in alcohols, aceton or in their mixture under 0-50'C for a period of about one quater of an hour to about 24 hours. Preferably, this reaction is conducted in alcohol at room temperature. The reaction mixture was diluted by water and solid 2-dithiocarbamoylbenzcarboxamide was filtered off. For the 10 next step it is possible to use crude product or to recrystallize it from a suitable organic solvent. 2-Dithiocarbamoylbenzcarboxamide was treated by light alkaly (e.g. Na 2

HPO

4 , NaHCO 3 , Na 2

CO

3 , etc) in water, alcohols or in a mixture of water/alcohol at a temperature of 50-100C for 2-36 hours. Preferably, this reaction is conducted in a mixture of 15 water/alcohol at 50-75'C for about 24 hours. When the reaction is complete, the 2-substituted-4H-1,3-benzothiazin-4-one is obtained by conventional recovery procedures, e.g. trituration with ethylacetate or dilution with water, filtration and recrystallization from a suitable organic solvent. 20 Method B This method proposes to use excess of metal dithiocarbamate as light alkaly in the benzothiazinone cyclization and not to isolate 2 dithiocarbamoylbenzcarboxamide. So, the starting 2- WO 2009/010163 PCT/EP2008/005142 -6 chlorobenzcarboxamides can be treated with a 1,2-2,5 equimolar quantity of the metal salts of dithiocarbamates in alcohols, aceton or in their mixture at 20-80'C for a period of about 3-36 hours. Preferably, this reaction is conducted in alcohol or in a mixture of water/alcohol at 5 50-75 0 C for about 24 hours. The aimed 2-substituted-4H-1,3 benzothiazin-4-one is obtained by recovery procedures from method A. Method C

R

1 R1 R3 C1 KSC(S)OAlk R3 S OAIk R2* CONH 2 R2KC)N R4 R 4 0 R1 R5

HNR

5

R

6 S R6 R2 * 4 0 Method C 10 This method uses as starting material 2-chlorobenzencarboxamides. These compounds were treated with 1,1-2,0 fold excess of metal salts of alkylxantogenate, for example commercial available potassium ethylxantogenate, at a temperature of 20-100'C in different alcohols, acetone, acetonitrile or other suitable organic solvents for a period of is about one half hour to about 24 hours. Preferably, this reaction is conducted in alcohol at room temperature for about 24 hours. The isolated 2-alkoxy-4H-1,3-benzothiazin-4-one was treated with the corresponding amine HNR 5

R

6 in acetic acid, alcohols, ethylacetate, DMF, aceton or acetonitril for a period of up to 48 hours for full 20 exchange of the alkoxy group to the corresponding amine. After the process is completed the reaction mixture can be evaporated and diluted by water or it can be diluted by water directly. The aimed 2- NR 5

R

6 -4H 1,3-benzothiazin-4-one is recovered by customary isolation procedures, e.g. filtration and recrystallization from a suitable organic solvent. 25 -7 Method D The classical method of 1,3-benzothiazin-4-one synthesis by use of the reaction of thiocyanate salts with 2-chloroarylchloroanhydride and a subsequent treatment of the reaction mass with the corresponding amine is usable too. This method is well described in the scientific literature, for example: J. Imrich, P. Kristian, Coll. Czech. Chem. Commun., 1982, 47, 3268-3282; D. Koscik, P. Kristian, J. Gonda, E. Dandarova, Coil. Czech. Chem. Commun., 1983, 48, 3315-3328; D. Koscik, P. Kristian, 0. Forgac, Coll. Czech. Chem. Commun., 1983, 48, 3427-3432; T. H. Cronin, H. J .E. Hess, Pat. US 3522247. Surprisingly the compounds of the invention exhibit strong antibacterial activity, especially against mycobacteria with minimal inhibitory concentrations (MIC) in the range of <0,000012 - 0,78 pg/ml for fast growing mycobacteria, of <0.39 - 3.12 pg/ml for M tuberculosis, including multiresistant strains, determined by the classical method and of 2.0 - 50.0 ng/ml for M tuberculosis H37Rv determined by the Alamar Blue method. Surprisingly the compounds of the invention demonstrate a high level of selectivity for mycobacteria only which reduces the potential for adverse side effects dramatically. The compounds of the invention are non-mutagenic at 5 mg/ml in the SOS chromotest (M.Isidori, M. Lavorgna, A.Nardelli, L.Pascarella, A. Parella, Sci. Total Environ., 2005, 346, 87-98; M. Bombardier, N. Bermingham, R. Legault, A. Fouquet, Chemosphere, 2001, 42, 931-44; D.A. Widdick, D.I. Edwards, Mutat. Res., 1991, 259, 89-93). Thus, the compounds of the invention are useful for the treatment of tuberculosis infections and other mycobacterial infections, in humans and in animals. Accordingly, the invention concerns pharmaceutical compositions comprising a compound of the formula I of the invention. Also described is a compound of the formula I for use in a method for the treatment of bacterial infections in mammals.

WO 2009/010163 PCT/EP2008/005142 -8 Preferred compounds of the formula I for use in such method are those specifically listed above. The compounds of the invention are formulated for use by preparing a 5 diluted solution or suspension in pharmaceutically acceptable aqueous, organic or aqueous-organic medium for topical or parenteral administration by intravenous, subcutaneous or intramuscular injection, or for intranasal application; or are prepared in tablet, capsule or aqueous suspension form with conventional excipients for oral administration or 10 as suppositorium. The compounds can be used in dosages from 0.00 1 - 1000 mg/kg body weight. The examples which follow in the subsequent experimental part serve to is illustrate the invention but should not be construed as a limitation thereof. The structures of the compounds of the invention were established by modes of synthesis and elementary analysis, and by nuclear magnetic 20 resonance and mass spectra. Embodiments Starting materials 25 Chemicals and solvents were purchased from Alfa-Aesar (GB) or from Aldrich Co. (Sigma-Aldrich Company, St-Louis, US) and were used in the synthesis without additional purification. Melting points were determined according to the BP procedure and are uncorrected (Electrothermal 9001, GB). If analyses are indicated only by the symbols 30 of the elements, analytical results are within ±0.3% of the theoretical values (Carlo-Erba 5500, Italy). NMR spectra were determined with a Varian Unity Plus 400 (USA). Shifts for 1 H NMR are reported in ppm downfield from TMS (6). Mass spectra were obtained using a Finnigan SSQ-700 (USA) instrument with direct injection. Reactions and purity of WO 2009/010163 PCT/EP2008/005142 -9 compounds were controlled by TLC using Silicagel 60 F 254 aluminium sheets (Merck Co, Germany). Example 1 5 2-[4-(4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3-benzothiazin-4 one, (compound 1) 0.5 g of 2-chloro-3,5-dinitrobenzcarboxamide was dissolved in 25 ml 10 ethanol. The reaction mixture was treated with of 0.39 g of 4-(4 chlorophenyl)-piperazine dithiocarbamate sodium salt dihydrate and stored for 6 h at room temperature. The reaction mixture was poured into 50 ml of cooled water and the resulting yellow precipitate was filtered off. Pure final product was obtained after recrystallization from ethanol. 15 2-Aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)piperazine- 1 carbodithioate is a light yellow crystalline solid. Yield 68 %. mp 178 180 0 C. MS m/z 481 (M*). Anal. Calcd. for C 18

HI

6 ClN 5 0 5

S

2 : C, 44.86; H, 3.35; N, 14.53; S, 13.31 Found: C, 44.71; H, 3.36; N, 14.62; S, 13.35 20 0.5 g of 2-aminocarbonyl-4,6-dinitrophenyl-4-(4-chlorophenyl)pipe razine-1-carbodithioate was dissolved in 25 ml ethanol. The reaction mixture was treated with of 0.2 g of Na 2

HPO

4 x 12H 2 0 and refluxed for 6 h. Reaction mixture was cooled in the refrigerator and the ligth yellow 25 precipitate was filtered off and washed with 50 ml water and 30 ml methanol. Pure final product was obtained after recrystallization twice from ethanol. 2-(1,4-2-[4-(4-Chlorophenyl)piperazin-1-yl]-6,8-dinitro 4H-1,3-benzothiazin-4-one is a light yellow crystalline solid. Yield 38 %. mp 279-281 C (EtOH) 30 MS m/z 447 (M*). 'H NMR (DMSO-d 6 /CDCl 3 ) 6 9.08 and 8.95 (two 1H, two s, 2CH), 6.88 and 6.71 (two 2H, d, CH 4 Cl), 3,68 and 3.30 (two 4H, m,

N(CH

2

CH

2

)

2 N) ppm. Anal. Calcd. for C 18

H

1 4 ClN 5 0 5 S: C, 48.27; H, 3.15; N, 15.04; S, 7.16 35 Found: C, 48.34; H, 3.22; N, 14.97; S, 7.23 WO 2009/010163 PCT/EP2008/005142 -10 Example 2 2-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluorome thyl)-4H-1,3-benzothiazin-4-one, (compound 2) Following the procedure of Example 1 using of 2-chloro-3-nitro-5 5 trifluoromethylbenzcarboxamide as starting material. Light yellow crystalline solid. Yield 44%. mp 158-161'C (DMF/water) MS m/z 484 (Me). 1H NMR (DMSO-d/CDCl 3 ) 6 8.80 and 8.77 (two 1H, two s, 2CH), 7.32 (1H, s, CH), 9.95 and 6.73 (two IH, d, two CH), 3,65 and 3.29 (two 4H, 10 m, N(CH 2 CH2) 2 N), 2.29 (3H, s, CH 3 ) ppm. Anal. Calcd. for C 2 0

H

16 ClF 3

N

4 0 3 S: C, 49.54; H, 3.33; N, 11.55; S, 6.61 Found: C, 49.45; H, 3.40; N, 11.47; S, 6.83 Example 3 15 8-Nitro-6-(trifluoromethyl)-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1 yl}-4H-1,3-benzothiazin-4-one, (compound 3) Following the procedure of Example 1 using of 2-chloro-3-nitro-5 trifluoromethylbenzcarboxamide as starting material. Light yellow crystaline solid. Yield 33%. mp 201-203'C (EtOH). 20 MS m/z 504 (M). H NMR (DMSO-d 6 /CDCl 3 ) 6 8.80 and 8.77 (two IH, two s, 2CH), 7.61 (1H, s, CH), 7.39 and 7.03 (two IH, d, two CH), 3,66 and 3.31 (two 4H, m, N(CH 2 CH2) 2 N) ppm. Anal. Calcd. for C 20 H1 4

F

6

N

4 0 3 S: C, 47.62; H, 2.80; N, 11.11; S, 6.36 25 Found: C, 47.74; H, 2.91; N, 11.29; S, 6.53 Example 4 2-[Benzyl(ethyl)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothia zin-4-one, (compound 4) 30 A suspension of 1.2 g 2-chloro-3-amino-5-trifluoromethyl benzcarboxamide in 45 ml ethanol was treated with 2.0 g of benzyl(ethyl)dithiocarbamate sodium salt dihydrate and refluxed for 14 h. The dark red reaction mixture was diluted with 70 ml of water, cooled in the refrigerator for 6 hours, the ligth yellow precipitate was filtered off 35 and washed with 50 ml ester. Pure final product was obtained after WO 2009/010163 PCT/EP2008/005142 - 11 column chromatography (hexane/aceton 3:1). 2-[Benzyl(ethyl)amino]-8 nitro-6-(trifluoromethyl)-4H-1,3-benzothia-zin-4-one is a light yellow crystalline solid. Yield 40 %. mp 94-97C. MS m/z 409 (M*). 5 'H NMR (DMSO-dd/CDCl 3 ) 8 8.80 and 8.77 (two 1H, two s, 2CH), 7.41-7.25 (5H, m, C 6

H

5 ), 4.62 (2H, s, CH 2 ), 3.43 (2H, q, CH 2 ), 1.01 (3H, t, CH 3 ) ppm. Anal. Calcd. for C 18

H

14

F

3

N

3 0 3 S: C, 52.81; H, 3.45; N, 10.26; S, 7.83 Found: C, 52.73; H, 3.38; N, 10.44; S, 7.89 10 Example 5 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothia zin-4-one, (compound 5) Following the procedure of Example 4. Light yellow crystalline solid. is Yield 47%. mp 120-124C (EtOH/water). MS m/z 395 (M*). 'H NMR (DMSO-dr/CDCl 3 ) 5 8.81 and 8.77 (two 1H, two s, 2CH), 7.40-7.25 (5H, m, C 6

H

5 ), 4.64 (2H, s, CH 2 ), 2.87 (3H, s, CH 3 ) ppm. Anal. Calcd. for C 17

H

12

F

3

N

3 0 3 S: C, 51.64; H, 3.06; N, 10.63; S, 8.11 20 Found: C, 51.76; H, 3.13; N, 10.41; S, 8.34 Example 6 2-[4-(2-Fluorophenyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3 benzothiazin-4-one, (compound 6) 25 Following the procedure of Example 1. Light yellow crystalline solid. Yield 37%. mp 164-168C (i-PrOH). MS m/z 454 (M*). 'H NMR (DMSO-d 6 /CDCl 3 ) 6 8.81 and 8.77 (two 1H, two s, 2CH), 6.76 (3H, m, 3CH), 6.11 (1H, m, CH), 3,68 and 3.30 (two 4H, m, 30 N(CH 2

CH

2

)

2 N) ppm. Anal. Calcd. for Cj 9

H

14

F

4

N

4 0 3 S: C, 50.22; H, 3.11; N, 12.33; S, 7.06 Found: C, 50.08; H, 3.21; N, 12.46; S, 7.09 Example 7 35 2-(4-Benzylpiperazin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzo thiazin-4-one, (compound 7) WO 2009/010163 PCT/EP2008/005142 - 12 Following the procedure of Example 4. Yellow crystalline solid. Yield 51%. mp 161-163'C (EtOH/DMF). MS m/z 450 (M*). 'H NMR (DMSO-d6/CDCl 3 ) 5 8.80 and 8.76 (two 1H, two s, 2CH), 5 7.19-7.28 (5H, m, Ph), 3.48 (2H, s, CH 2 ), 3,38 and 3.09 (two 4H, m,

N(CH

2

CH

2

)

2 N) ppm. Anal. Calcd. for C1 7

H

18

N

4 0 7 S: C, 53.33; H, 3.80; N, 12.44; S, 7.12 Found: C, 53.29; H, 4.01; N, 12.48; S, 7.06 10 Example 8 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, (compound 8) A suspension of 2.5 g 2-chloro-3 -nitro-5 -trifluoromethylbenzcar 15 boxamide in 25 ml ethanol was treated with of 1.75 g of sodium ethylxantogenate and stored for 24 h at room temperature. The reaction mixture was poured into 50 ml of cooled water and the resulting yellow precipitate was filtered off. Pure 2-ethoxy-8-nitro-6-(trifluoromethyl) 4H-1,3-benzothiazin-4-one was obtained after recrystallization from 20 ethanol/water as white crystalline solid. Yield 58 %. mp 146-148C. MS m/z 320 (M*). Anal. Calcd. for C,,H 7

F

3

N

2 0 4 S: C, 41.26; H, 2.20; N, 8.75; S, 10.01 Found: C, 41.34; H, 2.22; N, 8.87; S, 10.27 A solution of 0.7 g of 2-ethoxy-8-nitro-6-(trifluoromethyl)-4H-1,3 25 benzothiazin-4-one in 15 ml acetic acid was treated with 0.4 ml of benzylamine and refluxed for 14 h. The reaction mixture was evaporated and the residue was treated by 10 ml water, the yellow precipitate was filtered off and washed with 50 ml water. Pure final product was obtained after recrystallization twice from ethanol/DMF. 2 30 (Benzylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one is a light yellow crystalline solid. Yield 73 %. mp 192-194C. MS m/z 381 (M*). 'H NMR (DMSO-d 6 /CDCl 3 ) 5 9.27 (1H, broad s, NH), 8.80 and 8.75 (two 1H, two s, 2CH), 7.74-7.49 (5H, m, Ph), 4.49 (2H, s, CH 2 ) ppm.

WO 2009/010163 PCT/EP2008/005142 - 13 Anal. Calcd. for CiH 10

F

3

N

3 0 3 S: C, 50.39; H, 2.64; N, 11.02; S, 8.41 Found: C, 50.42; H, 2.61; N, 10.89; S, 8.64 Example 9 2- {Methyl[(1R)-1-phenylethyl]amino} -8-nitro-6-(trifluoromethyl)-4H 5 1,3-benzothiazin-4-one, (compound 9) Following the procedure of Example 1. Light yellow crystalline solid. Yield 54%. mp 110-113C (purification by column chromatography aceton/hexane 5:1). MS m/z 409 (M*). 10 'H NMR (DMSO-dr/CDCl 3 ) 6 8.82 and 8.76 (two 1H, two s, 2CH), 7.84, 7.43, 7.15 (5H, 3 m, Ph), 4.84 (H, m, CH), 3.07 (3H, s, NCH 3 ), 1.39 (3H, d, CH 3 ) ppm. Anal. Calcd. for C1 8

H

14

F

3

N

3 0 3 S: C, 52.81; H, 3.45; N, 10.26; S, 7.83 Found: C, 52.73; H, 3.46; N, 10.19; S, 7.92 15 Example 10 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one, (compound 10) Following the procedure of Example 8. Light yellow crystalline solid. 20 Yield 64%. mp 138-141'C (purification by column chromatography aceton/hexane 4:1). MS m/z 361 (Mi). 'H NMR (DMSO-dJ/CDCl 3 ) 6 8.37 and 8.23 (two 1H, two s, 2CH), 7.45-7.35 (5H, m, Ph), 4.62 (2H, 2, CH 2 ), 2.87 (3H, s, CH 3 ) ppm. 25 Anal. Calcd. for C 16

H

1 2 C1N 3 0 3 S: C, 53.11; H, 3.34; N, 11.61; S,8.86 Found: C, 53.19; H, 3.30; N, 11.52; S, 8.89 Example 11 Determination of the in vitro inhibitory activity of the compounds of the 30 invention against mycobacteria. The antibacterial activities of the compounds against Mycobacterium smegmatis SG 987, M. aurum SB66, M. vaccae IMET 10670 and M. fortuitum B were tested by determination of minimal inhibitory WO 2009/010163 PCT/EP2008/005142 -14 concentrations (MIC) by the broth micro dilution method in Mueller Hinton broth (Difco) according to the NCCLS guidelines [National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 5 th 5 Ed.; Villanova, Ed.; Approved standard Document M7-A5. NCCLS, (2000)]. The results are presented in Table 1. Table 1: Antimicrobial activity of compounds as of the formula I determined by minimal inhibitory concentrations MIC [pg/ml] 10 Compound M. smegmatis M.vaccae M.fortuitum MIC [tg/ml] MIC [tg/ml] MIC [pg/ml] 1 0,78 0,0062 0,78 2 0,05 0,008 0,031 3 0,04 0,0031 0,0031 4 0,0062 0,0008 0,0062 5 0,0156 0,0005 0,0156 6 0,005 0,005 0,005 7 0,05 0,05 0,05 8 0,00078 <0,0000121 0,00078 9 <0,0000121 <0,0000121 <0,0000121 10 0,0062 0,00031 0,00156 Example 12 Activity against M. tuberculosis H37Rv was tested by the following method for determination of minimal inhibitory concentrations (MIC) is and minimal bactericidal concentrations (MBC): Strains were inoculated onto solid Lowenstein-Jensen medium. After 21 days, the cultures grown were used to prepare an inoculum suspension corresponding to 5 x 108 microbial cells/ml. With 0,2 ml of that suspension tubes with 2 ml liquid Shkolnikova medium, containing 20 corresponding concentrations of compounds under study - from 100,0 to 0,195 [tg/ml, were inoculated. After 14 days of incubation at 37 C the tubes with liquid medium were centrifuged for 15min. at 3000 RPM. After discarding the supernatant, the sediment was resuspended in 0,8 ml of sterile 0,9% NaCl. 0,1 ml of the suspension was used to prepare 25 smears subsequently stained by the Ziehl-Neelsen method. The WO 2009/010163 PCT/EP2008/005142 - 15 remaining sediment was inoculated in 0,2 ml volumes into three tubes with solid drug free Lowenstein-Jensen medium to determine minimal bactericidal concentrations (MBC). The results were read after 21-28 days of cultivation at 37 C. Controls were tubes cultured with test 5 strains not treated with the studied agents. Minimal bactericidal concentration of drugs (MBC) was considered as the drug concentration completely inhibiting the growth of mycobacteria on the solid medium. The bacteriostatic effect (MIC) was characterized by the presence of only individual mycobacteria in the smear and a 10 strong decrease in the number of colonies grown on solid media compared to the controls. The results are presented in Table 2. Table 2: Antimicrobial activity of compounds of the formula I against Mycobacterium tuberculosis H37Rv and clinical isolates HSRE resistant strain and XTB strain as determined by minimal inhibitory concentrations 15 (MIC) and minimal bactericidal concentrations (MBC) Strain Compound MBC MIC (ptg/mL) (pg/mL) H37Rv 3.12 1.56 HSRE resistant 5 3.12 1.56 X TB 3.12 1.56 H37Rv 1.56 1.56 HSRE resistant 8 1.56 0.78 X TB 1.56 0.78 H37Rv 0.78 <0.39 HSRE resistant 10 1.56 1.56 X TB 0.39 <0.39 H37Rv 1.56 0.78 HSRE resistant Isoniazid (INH) Not active XTB Not active HSRE: multiresistant strain XTB: extremly resistant strain Example 13 Activity against M. tuberculosis H37Rv was determined by the resazurin 20 reduction assay (MIC 96 ) too. The method was described in detail in: P. Quillardet, 0. Huisman, R. D'Ari, M. Hofnung, Proc. Natl. Acad. Sci. USA, 1982, 79, 5971-5; J.C. Palomino, A. Martin, M. Camacho, H. Guerra, J. Swings, F. Portaels, Antimicrob. Agents Chemother., 2002, 46, 2720-2. The results are presented in Table 3. 25 - 16 Table 3: Antimycobacterial activity of compounds as of the formula I determined by minimal inhibitory concentrations (MIC) [ng/ml] Compound M. tuberculosis H37Rv (n/i 4 2 5 _3 8 50 0,4 10 25 INK 125 The term "comprising" as used in this specification and claims means "consisting at least in part of'. When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner. In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Claims

1. A compound of formula (I) R1 R5 R3 rS NR R2 N R 4 0 (I) or a salt thereof, wherein R 1 and R2 are independently from each other NO 2 , NR 7 R', NHOR 9 , COOR 9 , CN, CONR' 0 R", CHO, F, Cl, Br, SO 2 NR 2 R 13 , lower alkoxy, OCF 3 , mono-, di or trifluoromethyl; R3 and R4 are independently from each other II, a saturated or unsaturated, linear or branched aliphatic radical having 1-3 chain members, F, Cl, Br, or lower alkoxy; Rs is H, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members; R 6 is a radical: R14 wherein X is a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, or RIs and l 6 together represent bivalent radicals: or R5 18 wherein n is 1-4; R7-R"3 are independently from each other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-5 chain members, phenyl, benzyl or R7 and R 8 together, R' 0 and R" together, R1 2 and R 13 together represent a linear or branched aliphatic bivalent radical having 1-7 chain members; R1 4 and R1 5 are independently from each other H, a linear or branched aliphatic radical having 1-5 chain members, F, Cl, Br, NO 2 , NH 2 , or CF 3 2. A compound according to formula (I) of claim 1, selected from the group consisting of: A) 2-[4-(2-R", 5-R' 5 -Phenyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)-4H-1,3 benzothiazin-4-one, wherein R 4 represents H and R 5 represents F, Cl, or Br, B) 2-[4-(3-R1 4 , 5-R 5 -Phenyl)piperazin-1 -yl]-8-nitro-6-(trifluoromethyl)-4H-1,3 benzothiazin-4-one, wherein R1 4 represents H and R1 5 represents F or Cl, C) 2-[Benzyl(methyl)amino]-8-nitro-6-R 2 -4H-1,3-benzothiazin-4-one, wherein R2 represents CF 3 , Cl or F. D) 2-[Benzyl(R 5 )amino]-8-R' -6-(trifluoromethyl)-4H- 1,3-benzothiazin-4-one, wherein R 1 represents CF 3 , NO 2 , NI 1011 or NR 7 R 8 and Rs, R and R 8 have the same meaning as defined in claim 1,. E) 6-R 2 -2-[Methyl(2-phenylethyl)amino]-8-nitro-4H-1,3-benzothiazin-4-one, wherein R 2 has the same meaning as defined in claim 1, F) 6-Trifluoromethyl-2-[methyl(2-phenylethyl)amino]-8-R'-4H-1,3-benzothiazin

4-one, wherein R' has the same meaning as defined in claim 1, G) 2-[4-(2-R1 4 , 6-R' 5 -Phenyl)piperazin-1-yl]-8-nitro-6--(trifluoromethyl)-4H-1,3 benzothiazin-4-one, wherein R1 4 and R 15 have the same meaning as defined in claim 1, H) 2-[Benzyl(Rs)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, wherein R 5 has the same meaning as defined in claim 1, I) 2-[Benzyl(R 5 )amino]-8-nitro-6-R2-4H-1,3-benzothiazin-4-one, wherein R2 and R5 have the same meaning as defined in claim 1, J) 2-[Benzyl(Rs)amino]-8-nitro-6-chloro-4H-1,3-benzothiazin-4-one, wherein R 5 has the same meaning as defined in claim 1, K) 2-[Benzyl(R 5 )amino]-8-nitro-6-fluoro-4H-1,3-benzothiazin-4-one according to formula (I) of claim 1, wherein R 5 has the same meaning as defined in claim 1. 3. A compound according to formula (I) of claim 1, selected from the group consisting of: 19 2- (Methyl[(1 R)- I -phenylethyl)amino } -8-nitro-6-(trifluoromethyl)-4H-1,3 benzothiazin-4-one, 2-[4-(4-Chlorophenyl)piperazin- I -yl]-6,8-dinitro-4H- 1,3-benzothiazin-4-one, 2-[4-(5-chloro-2-methylphenyl)piperazin- I -yl]-8-nitro-6-(trifluoromethyl)-4H- 1,3 benzothiazin-4-one,

8-Nitro-6-(trifluoromethyl)-2- { 4-[3-(trifluoromethyl)phenyl]piperazin- 1-yl} -4H- 1,3 benzothiazin-4-one, 2-[Benzyl(ethyl)amino] -8-nitro-6-(trifluoromethyl)-4H- 1,3 -benzothiazin-4-one, 2-[Benzyl(methyl)amino]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2-[4-(2-Fluorophenyl)piperazin- 1-yl]-8-nitro-6-(trifluoromethyl)-4H- 1,3-benzothiazin 4-one, 2-(4-Benzylpiperazin- I -yl)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2-(Benzylamino)-8-nitro-6-(trifluoromethyl)-4H1-1,3-benzothiazin-4-one, and 2-[Benzyl(methyl)amino]-6-chloro-8-nitro-4H- 1,3-benzothiazin-4-one, or a salt thereof. 4. Use of a compound of formula (I) or a salt thereof according to any of the preceding claims for the preparation of a pharmaceutical composition. 5. Use of a compound of formula (I) or a salt thereof according to any of the preceding claims for the preparation of a medicament for the therapeutic or prophylactic treatment of microbial infection in mammals. 6. Use according to claim 5, wherein the microbial infection in mammals is a tuberculosis or leprosy infection. 7. Pharmaceutical composition comprising a compound according to any of the preceding claims. 8. A compound according to any of the preceding claims or a salt thereof for use in a method for the therapeutic or prophylactic treatment of microbial infection in mammals.

9. A compound according to any of the preceding claims or a salt thereof for use in a method for the therapeutic or prophylactic treatment of tuberculosis or leprosy infection in mammals.

10. Method for the preparation of 2-NR 5 R 6 -4H-1,3-benzothiazin-4-ones according to formula (I) of claim 1, comprising reacting 2-chlorobenzcarboxamides with 1.1 to 2.5 times in 20 excess of metal salts of alkylxanthogenate at a temperature of 20-1 00"C in a solvent and subsequently treating the resulting 2-alkoxy-4H-1,3-benzothiazin-4-one with an amine HNR 5 R 6 in a solvent at a temperature of 20-100 C.

11. A compound as defined in claim I substantially as herein described with reference to any example thereof.

12. A use as defined in any one of claims 4 to 6 substantially as herein described with reference to any example thereof.

13. A pharmaceutical composition as claimed in claim 7 substantially as herein described with reference to any example thereof.

14. A compound as claimed in claim 8 or claim 9 substantially as herein described with reference to any example thereof.

15. A method as claimed in claim 10 substantially as herein described with reference to any example thereof.